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Abstract:

The present invention relates to a specimen box for an electron
microscope, comprising a first substrate, a second substrate, one or more
photoelectric elements, and a metal adhesion layer. The first substrate
has a first surface, a second surface, a first concave, and one or more
first through holes, wherein the first through holes penetrate through
the first substrate. The second substrate has a third surface, a forth
surface, and a second concave. The photoelectric element is disposed
between the first substrate and the second substrate. In addition, the
metal adhesion layer is disposed between the first substrate and the
second substrate to form a space for a specimen contained therein.
Besides, the present specimen box further comprises one or more plugs.
When the plugs are assembled into the first through holes to seal the
specimen box, the in-situ observation can be accomplished by using the
electron microscope.

Claims:

1. A specimen box for an electron microscope, comprising: a first
substrate, which has a first surface, a second surface, a first concave,
and one or more first through holes, wherein the first concave is
disposed on the second surface, a first thin film corresponding to the
first concave is disposed on the first surface, and the first through
hole is disposed around the first concave and penetrates through the
first substrate; a second substrate, which has a third surface, a fourth
surface, and a second concave, wherein the second concave is disposed on
the fourth surface, and a second thin film corresponding to the second
concave is disposed on the third surface; a metal adhesion layer, which
is disposed between the first substrate and the second substrate; and one
or more photoelectric elements, which comprises one or more ends, and the
photoelectric element is disposed between the first substrate and the
second substrate; wherein a space is formed by the first substrate, the
second substrate, and the metal adhesion layer, and the end is disposed
in the space.

2. The specimen box for an electron microscope as claimed in claim 1,
wherein the first through hole penetrates through the first thin film.

3. The specimen box for an electron microscope as claimed in claim 1,
wherein the metal adhesion layer is disposed between the second surface
and the fourth surface.

4. The specimen box for an electron microscope as claimed in claim 1,
wherein the metal adhesion layer is disposed between the second surface
and the second thin film.

5. The specimen box for an electron microscope as claimed in claim 1,
wherein the photoelectric element is independently a photo fiber or an
electrode.

6. The specimen box for an electron microscope as claimed in claim 1,
wherein the second substrate further comprises one or more second through
holes, wherein the second through hole is disposed around the second
concave and penetrates through the second substrate.

7. The specimen box for an electron microscope as claimed in claim 1,
wherein the hole size of the first through hole is 10 μm to 1000
μm.

8. The specimen box for an electron microscope as claimed in claim 6,
wherein the hole size of the second through hole is 10 μm to 1000
μm.

9. The specimen box for an electron microscope as claimed in claim 1,
wherein the specimen box further comprises one or more plugs assembled
into the first through holes.

10. The specimen box for an electron microscope as claimed in claim 6,
wherein the specimen box further comprises one or more plugs assembled
into the second through holes.

11. The specimen box for an electron microscope as claimed in claim 1,
wherein the material of the first thin film and the second thin film is
independently silicon dioxide (SiO2), silicon nitride
(Si3N4), or a combination thereof.

12. The specimen box for an electron microscope as claimed in claim 1,
wherein the thickness of the first thin film and the second thin film is
independently 1 nm to 100 nm.

13. The specimen box for an electron microscope as claimed in claim 1,
wherein a first protective layer is disposed on the surface of the first
thin film.

14. The specimen box for an electron microscope as claimed in claim 13,
wherein the material of the first protective film is silicon nitride
(Si3N4).

15. The specimen box for an electron microscope as claimed in claim 1,
wherein a second protective layer is disposed on the surface of the
second thin film.

16. The specimen box for an electron microscope as claimed in claim 15,
wherein the material of the second protective film is silicon nitride
(Si3N4).

17. The specimen box for an electron microscope as claimed in claim 1,
wherein the material of the first substrate and the second substrate is
independently silicon substrate, glass substrate, or polymer substrate.

18. The specimen box for an electron microscope as claimed in claim 1,
wherein the thickness of the first substrate and the second substrate is
independently 10 μm to 1000 μm.

19. The specimen box for an electron microscope as claimed in claim 1,
wherein the metal adhesion layer comprises a metal material, wherein the
metal material is selected from a group consisting of Ti, Cr, Sn, In, Bi,
Cu, Ag, Ni, Zn, Au, and Ti--W alloy.

20. The specimen box for an electron microscope as claimed in claim 1,
wherein the material of the plugs is selected from a group consisting of
Ni--Ti alloy, copper-base alloy, Cu--Zn alloy, Cu--Al--Mn alloy,
Cu--Al--Ni alloy, Cu--Al--Be alloy, Cu--Al--Be--Zr alloy, and
Cu--Al--Ni--Be alloy.

21. The specimen box for an electron microscope as claimed in claim 1,
wherein the volume of the space is 0.01 mm3 to 100 mm.sup.3.

Description:

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefits of the Taiwan Patent
Application Serial Number 100123732, filed on Jul. 5, 2011, the subject
matter of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to specimen box with one or more
through holes for use with an electron microscope, especially to a
specimen box with one or more photoelectric elements for use with an
electron microscope.

[0004] 2. Description of Related Art

[0005] As known in the prior art, a vacuum environment inside an electron
microscope is critical for high resolution and accuracy during the
observation of specimens under the electron microscope. Considering the
vacuum environment, the conventional electron microscope is usually used
to observe the structures of solid substances or specimens, such as
dehydrated bio-tissueor dehydrated virus. Hence, the conventional
electron microscope has a limitation on the selection of specimens and is
invalid for the dynamic observation of specimens. Even the
stimuli-induced responses of specimens are unobservable under the
conventional electron microscope.

[0006] Due to the above-mentioned limitations, the application of the
electron microscope is restricted. In order to improve the
above-mentioned drawbacks, a specimen box for an electron microscope
suitable for the observation of specimens (such as chemical particles,
biochemical molecules, or bio-tissues) in a gas or liquid state was
proposed. After the specimen is injected into this specimen box, a
sealant or a polymer sealant is used for sealing the specimen box.
However, the vacuum degree of the electron microscope may be degraded due
to the easy evaporation of liquid from the specimen into the vacuum
environment through the sealant or the polymer sealant. Accordingly, the
resolution and the observation efficiency of the electron microscope
would be greatly affected by the above-mentioned factors.

[0007] Another specimen box for an electron microscope was also suggested
to solve the above issues. The specimen box further includes a gas
chamber in addition to the specimen chamber. Accordingly, the gas
diffusion or the liquid evaporation from the specimens in the specimen
chamber can be inhibited by the pressure equilibrium between the specimen
chamber and the gas chamber. However, the inert gas filled in the gas
chamber for the pressure equilibrium may affect the observation
resolution. In addition, the structure of this specimen box is
complicated so the cost is increased.

[0008] In all of the current and above specimen boxes, none could be
opened again (=reopen) after the specimen boxes are sealed. Due to finite
oxygen contained in the closed space, the long-term dynamic changes and
the light- or current-induced responses of the specimens cannot be
observed under the electron microscope and thus the observation of living
tissues or cells is limited.

[0009] According to above, providing a specimen box, which has high
sealing and reopening features and allows the application of ambient
stimuli (such as light, or current), is advantageous to the long-term
dynamic observation of specimens in a gas or liquid state and the
variation of the observation condition under an electron microscope.

SUMMARY OF THE INVENTION

[0010] The object of the present invention is to provide a specimen box
for an electron microscope, in which the specimen box comprises through
holes and plugs. Hence, the specimen box can be reopened to inject gas or
liquid again so as to prolong the in-situ observation time effectively.

[0011] Another object of the present invention is to provide a specimen
box with photoelectric elements. Hence, when the specimen is stimulated
by light or current by the photoelectric elements, the observation of
dynamic changes and response of the specimen could be obtained.

[0012] To achieve the object, the current specimen box for an electron
microscope comprises: a first substrate, a second substrate, a metal
adhesion layer, and one or more photoelectric elements. The first
substrate has a first surface, a second surface, a first concave, and one
or more first through holes, wherein the first concave is disposed on the
second surface, a first thin film corresponding to the first concave is
disposed on the first surface, and the first through hole is disposed
around the first concave and penetrates through the first substrate. The
second substrate has a third surface, a fourth surface, and a second
concave, wherein the second concave is disposed on the fourth surface,
and a second thin film corresponding to the second concave is disposed on
the third surface. The metal adhesion layer is disposed between the first
substrate and the second substrate. In addition, a space, which is formed
by the first substrate, the second substrate, and the metal adhesion
layer, could contain the gas or liquid specimens in the specimen box. The
photoelectric element comprises one or more ends, wherein the
photoelectric element is disposed between the first substrate and the
second substrate, and the end is disposed in the space. The specimen used
for the specimen box, such as chemical atoms, molecules, complexes,
mixtures, bio-tissues, cells, enzymes, nerve cells, photoreceptor cells,
or stem cells is not particularly limited as long as it could be observed
by the electron microscope. The photoelectric element can transmit light
or transfer current to the end of the photoelectric element. Therefore,
the dynamic change and response of the specimen could be observed by the
stimuli of light or current.

[0013] According to the specimen box of the present invention, the first
through hole penetrates through the first thin film and the first
substrate to connect the space in the specimen box with the outside
space. Therefore, the specimen box could be opened by the first through
hole, and the specimens, gas, or liquid could be inserted or injected
into the specimen box through the first through hole.

[0014] In the present invention, the first concave and the second concave
are formed by photolithography process accompanied by wet etching
process, dry etching process, or deep reactive-ion etching process. The
shape of the first concave and the second concave could be regular shape
or irregular shape. Preferably, the shape of the first concave and the
second concave is independently a cylinder, a cone, a cube, or a cuboid.

[0015] In the present invention, one or more third concave are disposed on
the second surface of the first substrate, and one or more fourth concave
are disposed on the fourth surface of the second substrate. One or more
photoelectric elements are disposed on the third concave and the fourth
concave. The photoelectric element could transmit light or transfer
current to the end. The end could release light or current in the space.
Therefore, the light or current could stimulate the specimen in the
space. The third concave and the fourth concave are formed by
photolithography process accompanied by wet etching process, dry etching
process, or deep reactive-ion etching process, particularly, are formed
by photolithography process accompanied by wet etching process. The
disposition of the third concave and the fourth concave are not
especially limited. The preferable position of the third concave and the
fourth concave is at the diagonal position of the first substrate and the
second substrate. The photoelectric element is independently a
photoelectric conversion element, an optical element, or an electric
element. Preferably, the photoelectric element is independently an
optical fiber, or an electrode. The optical fiber is a gradient fiber, a
multi-mode mutant fiber, a single mode fiber, a multi-mode fiber, a
photonic crystal fiber, or etc. In the present invention, the
photoelectric element could be disposed, sealed, and fixed on the third
concave and the fourth concave by O-ring, sealant, polymer sealant,
solder, or etc.

[0016] The metal adhesion layer of the specimen box could be disposed
between the second surface and the fourth surface, the second surface and
the second thin film, or the first thin film and the second thin film, in
order to form a space with different volume and shape. Hence, according
to the different specimen volume and different observed resolution, the
volume and the shape of the space could be adjusted by the disposition of
the metal adhesion layer. The volume of the space is 0.01 mm3 to 100
mm3. Preferably, the volume of the space is 0.05 mm3 to 50
mm3. Most preferably, the volume of the space is 0.1 mm3 to 10
mm3. The height of the space is between 10 μm to 1000 μm.
Preferably, the height of the space is between 20 μm to 700 μm.
Most preferably, the height of the space is between 30 μm to 550
μm. The material of the metal adhesion layer preferably comprises a
metal material to form a solder, in which the metal material is selected
from a group consisting of Ti, Cr, Sn, In, Bi, Cu, Ag, Ni, Zn, Au, and
Ti--W alloy. Preferably, the metal material is Sn, Ni, Zn, Au, In, or a
combination thereof. Most preferably, the metal material is Sn, Au, or a
combination thereof. In addition, the metal adhesion layer could further
comprise an adhesion layer, a metallurgy layer, and a solder layer, in
which the material of the adhesion layer is Ti, Ti--W alloy, or Cr; and
the material of the metallurgy layer is Ni, Cu, or Au. The metal material
used in the present invention has excellent features of waterproofing,
high sealing, and biocompatibility. However, the material of the metal
adhesion layer has to be heated to a high temperature so as to allow the
upper substrate and the lower substrate to adhere together. The high
temperature thus may destroy the specimen in the specimen box. Hence, the
preferably method to solve this problem is that the metal adhesion layer
adheres the first substrate and the second substrate together at
70° C., then the specimen is inserted or injected into the
specimen box. Therefore, the specimen would not be destroyed by the high
temperature.

[0017] In the present invention, the second substrate could further
comprise one or more second through holes, in which the second through
hole is disposed around the second concave and penetrates through the
second substrate. Therefore, the space of the specimen box could connect
with the outside space, and the specimen, gas, and liquid could be
inserted or injected into the space through the second through hole.

[0018] The hole size of the mentioned first through hole is 10 μm to
1000 μm. Preferably, the hole size of the first through hole is 50
μm to 700 μm. Most preferably, the hole size of the first through
hole is 100 μm to 500 μm. In addition, the hole size of the
mentioned second through hole is 10 μm to 1000 μm. Preferably, the
hole size of the second through hole is 50 μm to 700 μm. Most
preferably, the hole size of the second through hole is 100 μm to 500
μm. The hole size of the first through hole and the second through
hole could be adjusted according to the different requirements for
observation. The method for forming the first through hole and the second
through hole is preferably a deep reactive-ion etching process or laser
drilling process. In fact, the first through hole and the second through
hole are passages to inject a gas specimen or a liquid specimen.
Additionally, the first through hole and the second through hole also
could be used to inject gas or liquid such as oxygen, nitrogen, buffer,
or medium, as is required by the specimen. Therefore, the observation
time of the specimen could be prolonged. For example, if oxygen and
medium are injected into the space via the through hole, the lifetime of
the cell specimen in the specimen box could not only be prolonged, but
also the in-situ observation time is prolonged. The dynamic changes of
the cell specimen could therefore also be observed. In the present
invention, the specimen box for an electron microscope of the present
invention could further comprise one or more plugs assembled into the
first through holes and the second through holes. The material of the
plug is not especially limited, which could be metal, memory metal,
polymer, plastic, ceramic, acrylic, or a combination thereof. Preferable,
the material of the plug is memory metal, polymer, plastic, ceramic, or a
combination thereof. Most preferably, the material of the plug is memory
metal. Then, the material of memory metal could select from a group
consisting of Ni--Ti alloy, copper-base alloy, Cu--Zn alloy, Cu--Al--Mn
alloy, Cu--Al--Ni alloy, Cu--Al--Be alloy, Cu--Al--Be--Zr alloy, and
Cu--Al--Ni--Be alloy. Preferably, the material of memory metal is Ti--Ni
alloy, Cu--Zn alloy, Cu--Al--Ni alloy, or a combination thereof. Most
preferably, the material of memory metal is Ni--Ti alloy. Because memory
metal has a property of thermal expansion and contraction, the plugs of
the present invention preferably are used for sealing the through hole,
and a tight sealing of the specimen box in the present invention could be
accomplished.

[0019] In the specimen box for an electron microscope of the present
invention, the material of the first thin film and the second thin film
is independently silicon dioxide (SiO2), silicon nitride
(Si3N4), or a combination thereof. The function of the first
thin film and the second thin film is increasing the selectivity in the
etching process and enhancing the hardness of the surface of the first
substrate and the second substrate. In addition, the thickness of the
first thin film and the second thin film is independently 1 nm to 100 nm.
Preferably, the thickness of the first thin film and the second thin film
is independently 5 nm to 80 nm.

[0020] In the present invention, the specimen box further comprises a
first protective layer on the surface of the first thin film, in which
the first protective layer is disposed on the surface of the first thin
film. Similarly, the specimen box also comprises a second protective
layer on the surface of the second thin film, in which the second
protective layer is disposed on the surface of the second thin film.
Preferably, the material of the first protective layer and the second
protective layer is silicon nitride (Si3N4), in which silicon
nitride (Si3N4) is hard enough to protect the first thin film
and the second thin film, and could prevent cracking of the first thin
film and the second thin film. Furthermore, the first protective layer
and the second protective layer could increase the selectivity in the
etching process.

[0021] In the specimen box of the present invention, the first substrate
and the second substrate is independently silicon substrate, glass
substrate, or polymer substrate. Preferably, the first substrate and the
second substrate is silicon substrate. In addition, the thickness of the
first substrate and the second substrate is independently about 10 μm
to 1000 μm. Preferably, the thickness of the first substrate and the
second substrate is independently about 100 μm to 250 μm.

[0022] According to above, before the specimen is injected in the specimen
box, the photoelectric elements are disposed, fixed, and sealed on the
third concave and the fourth concave by O-ring, sealant, polymer sealant,
or solder, preferably by solder. Then, the metal adhesion layer adheres
the first substrate and the second substrate together at high
temperature. Besides, the specimen box comprises the first through hole
and the second through hole, which are passages to insert or inject the
specimen into the space of the specimen box. When the specimen is
inserted or injected into the specimen box, the plugs, especially the
memory metal plugs, would be assembled into the first through holes and
the second through holes to tightly seal the specimen box by the feature
of thermal expansion and contraction of the plugs. Therefore, the
specimen was totally sealed in the specimen box so as to be observed
through the electron microscope. When the specimen box is removed from
the electron microscope, the plugs could be removed to reopen the
specimen box temporarily. Therefore, a gas or liquid, as may be required
by the specimen, could additionally be injected into the specimen box to
prolong the lifetime of the specimen.

[0023] In addition, the manufacturing method of the specimen box in the
present invention is less complicated than the prior art, and the
materials of the specimen box of the present invention are also easy to
obtain. According to the above improvements of the specimen box in the
present invention, the kinds of specimens which could be observed by an
electron microscope are increased. After the specimen is stimulated by
the photoelectric elements, the in-situ observation of the specimen could
be obtained by using the specimen box of the present invention.

[0024] Other objects, advantages, and novel features of the invention will
become more apparent from the following detailed description when taken
in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 is a three-dimensional view showing the specimen box for an
electron microscope of the example 1;

[0026]FIG. 2 is a three-dimensional view showing the photoelectric
element of the specimen box for an electron microscope;

[0027]FIG. 3 is a perspective view showing the specimen box for an
electron microscope of the example 1;

[0028]FIG. 4 is a perspective view showing the specimen box for an
electron microscope of the example 2;

[0029]FIG. 5 is a perspective view showing the specimen box for an
electron microscope of the example 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0030] The present invention has been described in an illustrative manner,
and it is to be understood that the terminology used is intended to be in
the nature of description rather than of limitation. Many modifications
and variations of the present invention are possible in light of the
above teachings. Therefore, it is to be understood that within the scope
of the appended claims, the invention may be practiced otherwise than as
specifically described.

Example 1

[0031] As showing in FIG. 1, FIG. 2, and FIG. 3, FIG. 1 a
three-dimensional view showing the specimen box for electron microscope
of the example 1; FIG. 2 is a three-dimensional view showing the
photoelectric element of the specimen box for an electron microscope; and
FIG. 3 is a three-dimensional view, which is shown along the A-A' section
line in FIG. 1, showing the specimen box for an electron microscope of
the example 1. According to FIG. 1, FIG. 2, and FIG. 3, the specimen box
of the present invention comprises: a first substrate 11, a second
substrate 12, a metal adhesion layer 13, and four photoelectric elements
16. In the present example, the first substrate 11 and the second
substrate 12 are (001) silicon substrate. The thickness of the first
substrate 11 is 250 μm, and the thickness of the second substrate 12
is also 250 μm.

[0032] The first substrate 11 has a first surface 111, a second surface
112, a first concave 113, and two first through holes 114, in which the
first concave 113 is disposed on the second surface 112, and a first thin
film 111 corresponding to the first concave 113 is disposed on the first
surface 111. In addition, the first through holes 114 is disposed around
the first concave 113 and penetrates through the first substrate 11.

[0033] The second substrate 12 has a third surface 121, a fourth surface
122, and a second concave 123, in which the second concave 123 is
disposed on the fourth surface 122, and a second thin film 125
corresponding to the second concave 123 is disposed on the third surface
121.

[0034] Additionally, the metal adhesion layer 13 is disposed between the
second surface 112 of the first substrate 11 and the fourth surface 122
of the second substrate 12, in which a space (not shown) was formed by
the second surface 112, the fourth surface 122, and the metal adhesion
layer 13. Gas or liquid specimens could be contained in the space (not
shown). In the present example, the specimen is not especially limited as
long as the specimens could be observed by an electron microscope.

[0035] According to FIG. 1, FIG. 2, and FIG. 3, four fourth concaves 127
are formed on the diagonal position of the second surface 122 of the
second substrate, in which the fourth concaves 127 are formed along the
direction of <110> by photolithography process accompanied by the
wet etching process. The etching solution of the wet etching process is
NaOH solution. The shape of the fourth concave is V-shape. According to
FIG. 2, the photoelectric element 16 is disposed on each of the fourth
concave 127, and the end 161 of the photoelectric element 16 connects
with the space (not shown). Therefore, the specimen in the space (not
shown) could be stimulated by light or electricity.

[0036] The photolithography process and the wet etching process are used
for forming the first concave 113 on the second surface 112 and the
second concave 123 on the fourth surface 122. The shape of the first
concave 113 and the second concave 123 is cone.

[0037] In addition, the deep reactive-ion etching process is used for
forming the first through holes 114, in which the first through holes 114
penetrate through the first thin film 115 and the first substrate 11. The
hole size of the first through holes 114 is 250 μm.

[0038] The function of the first through holes 114 is as passages to
insert or inject the specimen into the space (not shown). Besides, the
first through holes 114 could also inject gas (such as oxygen, or
nitrogen) or liquid (such as buffer, acidic solution, or basic solution)
to further observe the dynamic changes of the specimen.

[0039] The material of the first thin film 115 and the second thin film
125 in the present example is SiO2, in which the function is
enhancing the hardness of the first substrate 11 and the second substrate
12 to avoid cracking of the substrates and increase the selectivity in
the etching process.

[0040] In the present example, the photoelectric element 16 is formed and
fixed on the fourth concave 127 by solder. The end 161 of the
photoelectric element 16 is connected with the space (not shown), the
other end of the photoelectric element 16 is extended out from the
specimen box, which connects with the source of light or electricity. In
the present example, the photoelectric element 16 is an optical fiber, an
electrode, or the combination of optical fiber and electricity.

[0041] The metal adhesion layer 13 in the present example comprises an
adhesion layer, a metallurgy layer, and a solder layer, in which the
material of the adhesion layer is Ti--W alloy, and the material of the
metallurgy layer is Cu. In the present example, the metal adhesion layer
13 adheres the first substrate 11 and the second substrate 12 to form the
space 14 by the method of automatic alignment packaging method at
150° C. After the first substrate 11 and the second substrate 12
are adhered together, the specimen would be inserted or injected into the
space (not shown) of the specimen box.

[0042] An electron beam from the electron microscope would penetrate
through the first concave 113 to the space (not shown) and penetrate
through the second concave 123. The volume of the space (not shown) is 4
mm3, and the height of the space 14 is 550 μm. In order to
enhance the hardness and etching selectivity of the substrate, a first
protective layer 116 is disposed on the surface of the first thin film
115, and a second protective layer 126 is disposed on the surface of the
second thin film 125. Furthermore, the material of the first protective
layer 116 and the second protective layer 126 is silicon nitride
(Si3N4).

[0043] Finally, the specimen box has two plugs 15, which could seal the
first through holes 114, to totally seal the specimen box. In addition,
the plugs 15 also could be removed from the first through holes 114,
therefore, the specimen box could be reopened according to the
requirement of the in-situ observation. The material of the plugs 15 is
Ti--Ni alloy. Because Ti--Ni alloy has the property of thermal expansion
and contraction, the volume of the plugs 15 is smaller below freezing
point than at room temperature. Therefore, when the plugs 15 below
freezing point are assembled in the first through holes 114 of the
specimen box with the room temperature, the volume of the plugs 15 would
gradually expand according to the gradually warming plugs 15. Afterwards,
the specimen box would be sealed completely as long as the first through
holes 114 are sealed by the plugs 15.

[0044] In the present example, a method of using a specimen box for
observing a living specimen is shown. First, the cell specimen is
inserted or injected in the space 14 through the first through holes 114.
The plugs 15 below freezing point are assembled in the first through
holes 114. After the temperature of the plugs 15 are warmed to room
temperature, the specimen box would be sealed. Then, the specimen box is
placed in the electron microscope to observe the cell specimen. In the
process of the observation, the specimen could be stimulated by light,
current, or the combination thereof, to complete the in-situ observation
of the dynamic changes and response of the specimen. According to the
requirements of the observation, one could further inject oxygen or
medium by removing and then replacing the plugs 15 to complete the
in-situ observation of the cell specimen.

Example 2

[0045]FIG. 4 is a perspective view showing the specimen box for an
electron microscope of the example 2. According to FIG. 4, the specimen
box of the present example is roughly the same as example 1. The only
difference is the disposition of the metal adhesion layer 13. In the
present example, the metal adhesion layer 13 is disposed between the
second surface 112 and the second protective layer 126. Four of the third
concaves 117 are formed on the second surface 112 of the first substrate
11 by a dry etching process. The photoelectric element 16, which is
disposed on the third concave 117, is an optical fiber, an electrode, or
the combination thereof. [0046] The volume of the space (not shown) in
the present example is 2 mm3. The height of the space (not shown) is
550 μm. In the present example, the volume of the space (not shown) is
smaller than example 1, so the resolution is higher than example 1.
Therefore, different volumes of the space (not shown) of the specimen box
could be chosen according to different requirements of the observation,
such as the volume of the specimen, and the required resolution.

Example 3

[0047]FIG. 5 is a perspective view showing the specimen box for an
electron microscope of the example 3. According to the FIG. 5, the
specimen box of present example is roughly the same as example 1. The
only difference in the present example is the first thin film 115, the
first protective layer 116, the second thin film 125, and the second
protective layer 126 are only disposed on the first concave 113 and the
second concave 123 to enhance the structure of the first concave 113 and
the second concave 123. Therefore, the first thin film 115 and the second
thin film 125 would not be cracked so as to avoid the specimen escaping
from the space (not shown).

[0048] A fourth concave 127 is formed on the second surface 122 of the
second substrate 12 by a wet etching process. The photoelectric element
16 is disposed on the fourth concave 127, in which the photoelectric
element 16 is an optical fiber, an electrode, or a combination thereof.

[0049] In addition, the first through holes 114 and the second through
holes 124 are formed by a deep reactive-ion etching process. Therefore,
the first through holes 114 are penetrated through the first surface 111,
and the second through holes 124 are penetrated through the third surface
121. The hole size of the first through holes 114 is 250 μm, and the
hole size of the second through holes 124 is also 250 μm. Finally, the
material of the plugs 15 in the present example is Ni--Ti alloy, and the
plugs 15 could also seal the first through holes 114 and the second
through holes 124 to seal the specimen box completely.

[0050] Although the present invention has been explained in relation to
its preferred embodiment, it is to be understood that many other possible
modifications and variations can be made without departing from the
spirit and scope of the invention as hereinafter claimed.